THE FULL ONCOGENIC ACTIVITY OF RET PTC2 DEPENDS ON TYROSINE-539, A DOCKING SITE FOR PHOSPHOLIPASE-C-GAMMA/

RET/PTC oncogenes, generated by chromosomal rearrangements in papillar y thyroid carcinomas, are constitutively activated versions of proto-R ET, a gene coding for a receptor-type tyrosine kinase (TK) whose ligan d is still unknown. RET/PTCs encode fusion proteins in which proto-RET TK and C-terminal domains are fused to different donor genes. The res pective Ret/ptc oncoproteins display constitutive TK activity and tyro sine phosphorylation. We found that Ret/ptcs associate with and phosph orylate the SH2-containing transducer phospholipase C gamma (PLC gamma ). Two putative PLC gamma docking sites, Tyr-505 and Tyr-539, have bee n identified on Ret/ ptc2 by competition experiments using phosphoryla ted peptides modelled on Ret sequence, Transfection experiments and bi ochemical analysis using Tyr-->Phe mutants of Ret/ptc2 allowed us to r ule out Tyr-505 and to identify Tyr-539 as a functional PLC gamma dock ing site in vivo. Moreover, kinetic measurements showed that Tyr-539 i s able to mediate high-affinity interaction with PLC gamma. Mutation o f Tyr-539 resulted in a drastically reduced oncogenic activity of Ret/ ptc2 on NIH 3T3 cells (75 to 90% reduction) both in vitro and in vivo, which correlates with impaired ability of Ret/ptc2 to activate PLC ga mma. In conclusion, this paper demonstrates that Tyr-539 of Ret/ptc2 ( Tyr-761 on the proto-RET product) is an essential docking site for the full transforming potential of the oncogene. In addition, the present data identify PLC gamma as a downstream effector of Ret/ptcs and sugg est that this transducing molecule could play a crucial role in neopla stic signalling triggered by Ret/ptc oncoproteins.